Abstracts

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy most often caused by SCN1A haploinsufficiency, which disrupts GABAergic interneuron function. Cell transplantation via medial ganglionic eminence (MGE) progenitors, the embryonic source of GABAergic interneurons, offers a mechanism-based therapeutic approach. Although rodent MGE transplants have shown promise in epilepsy models, the rabbit MGE hasn’t yet been characterized – a necessary step toward larger-animal translational studies. Rabbits represent a physiologically relevant intermediate model, featuring larger brains yet manageable gestation windows. Here, we present the first wild-type rabbit MGE identification at embryonic days E15, E18, and E21. We combined classic coronal Nkx2.1 mapping with a novel sagittal accessibility index and an immunohistochemical (IHC) panel. We hypothesized that E15 would yield the largest relative MGE dissection area, enriched in undifferentiated progenitors, thus representing the optimal harvest age for future transplantation.

Wild-type rabbit embryos were collected at E15, E18, and E21. Embryos were immersion-fixed, cryo-sectioned, and immunostained. Coronal sections spanning the rostrocaudal window – from the emerging lateral ventricle to the 4^th ventricle – were labeled for Nkx2.1 to define MGE length and cross-sectional area relative to total telencephalon. Sagittal sections were used to calculate lateral distance of the MGE from midline, yielding a computed accessibility index. Additional IHC quantified interneuron progenitor markers (Lhx6, Nrp1, Sox6), neuronal differentiation stage (Ki67, DCX, NeuN), and CGE contamination (Prox1/COUPTF2). Sections were imaged at low magnification to assess regional organization and at higher magnification to capture cellular details.

Across the three ages, the rabbit MGE exhibited a developmental pattern in which early-stage E15 tissue occupied a larger relative proportion of the telencephalon, displayed robust progenitor marker expression, and showed minimal neuronal differentiation or CGE overlap, all while remaining close to midline for straightforward dissection. By E18, the absolute Nkx2.1(+) domain reached its maximum size; however, this peak coincided with blurring boundaries as cells began to differentiate and migrate away from the MGE. By E21, the MGE had regressed further, progenitor markers had declined, and differentiation had increased substantially.

Although E18 offers the largest absolute Nkx2.1(+) domain, the emergence of migrating cells and loss of clear MGE boundaries reduce both cellular plasticity and harvest accessibility. By E21, further regression and ongoing migration compromises progenitor yield. In contrast, E15 provides the greatest relative MGE volume, highest levels of undifferentiated, proliferative markers, minimal CGE contamination, and closest proximity to midline – making it to the optimal timepoint for retrieving transplant-grade MGE tissue in wild-type rabbits. This work shows the first identification of rabbit MGE progenitors to guide future transplantation studies in models of DS and other epilepsies.